The HIV Nef protein is important for viral pathogenesis and progression to AIDS. Nef enhances HIV replication in resting CD4+ T cells and increases T cell activation in response to CD3/CD28 costimulation and other activating stimuli. The mechanisms by which Nef enhances viral replication in resting T cells and increases T cell activation in response to costimulatory signals or other activating stimuli are not known, however. HIV Nef association with p21-activated kinase 2 (Pak2) has been proposed to play a role in T cell activation, but whether Pak2 itself or another protein in the Nef-Pak2 complex is important for Nef-mediated effects on cell signaling/activation has not been determined. Furthermore, this question has been difficult to address experimentally because mutations in most Nef motifs previously reported to be required for Pak2 activation also affect other Nef functions such as CD4 or MHC-I downregulation. Preliminary studies identified primary HIV Nef clones that exhibit a similar capacity to downregulate CD4 and MHC-I but variable ability to associate with activated Pak2. By mutagenesis, we demonstrated that Nef amino acids at positions 85, 89, 187, 188 and 191 are critical for Pak2 association. Mutation of these Nef residues reduces or abolishes association with endogenous Pak2 activity but does not affect CD4 and MHC-I downregulation or virion infectivity. Mutation of 5C Nef residue 89 specifically abolish association with exogenous FLAG-tagged Pak2-K278R. Accordingly, these novel Nef mutants are valuable tools to identify and characterize cellular factors that bind directly to Nef to mediate its effects on T cell activation, and possibly other functions as well. Residues 85, 89, 187, 188 and 191 cluster on the surface of the Nef core domain in a region distinct from the dimerization and SH3-binding domains. We propose that these Nef residues form part of a unique binding surface specifically involved in association with Pak2 and other as-yet-unidentified protein-protein interactions to facilitate T cell activation, and that disrupting interaction of Nef with these proteins represents a potential therapeutic target. The overall goal of this application is to understand mechanisms by which Nef regulates T cell activation through interactions with Pak2 and other host cell proteins. We propose 3 specific aims: 1) Determine the functional requirement for Nef association with Pak2 in HIV replication in resting T cells and Nef-mediated enhancement of T cell activation;2) Identify cellular proteins associated with the HIV Nef-Pak2 complex and determine which protein in the complex interacts directly with Nef;3) Investigate the role of Nef and its association with active Pak2 in regulation of Cbl, an E3 ubiquitin ligase that negatively regulates T cell signaling. These studies will provide novel insights into functions of Nef and its interactions with cellular proteins, mechanisms by which Nef influences HIV replication and pathogenesis, and potential therapeutic approaches to inhibit Nef function.